Currently used methods of stable plant transformation usually employ direct (microprojectile bombardment, electroporation or PEG-mediated treatment of protoplasts, for review see: Gelvin, S. B., 1998, Curr. Opin. Biotechnol., 9, 227-232; Hansen & Wright, 1999, Trends Plant Sci., 4, 226-231) or Agrobacterium-mediated delivery of pre-engineered DNA fragment(s) of interest into plant cells. Manipulations with said DNA vectors in planta are restricted to simplifying the resolution of complex integration patterns (U.S. Pat. No. 6,114,600; Srivastava & Ow, 2001, Plant Mol Biol., 46, 561-566; Srivastava et al., 1999, Proc. Natl. Acad. Sci. USA, 96, 11117-11121) or removal of auxiliary DNA sequences from vectors stably integrated into chromosomal DNA. The methods of stable Agrobacterium-mediated integration of T-DNA regions within plant cells use whole desired DNA fragment flanked with left (LB) and right (RB) border sequences necessary for T-DNA transfer and integration into the host chromosomal DNA (U.S. Pat. No. 4,940,838; U.S. Pat. No. 5,464,763; EP0224287; U.S. Pat. No. 6,051,757; U.S. Pat. No. 5,731,179; WO9400977; EP0672752). In most cases, the approaches are directed to the integration of one specific T-DNA region into the chromosomal DNA. Also, co-integration of two or more different T-DNA regions was tried (U.S. Pat. No. 4,658,082). The latter approach is used for segregating different T-DNAs in progeny for various purposes. For example, Komari and colleagues (U.S. Pat. No. 5,731,179) describe a method of simultaneously transforming plant cells with two T-DNAs, one carrying a selectable marker functional in plants, while another T-DNA contains a desired DNA fragment to be introduced into plant cells.
In general, the DNA regions designed for stable integration into plant cells are pre-engineered in vitro by employing standard molecular biology techniques (Sambrook, Fritsch & Maniatis, 1989, Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor, N.Y.: CSH Laboratory Press). Also, in vivo engineering in bacterial cells is used, for example in order to assemble the binary vector with the help of homologous recombination (U.S. Pat. No. 5,731,179). Manipulations with T-DNA in planta are restricted to T-DNA regions pre-integrated into a chromosome like removing certain sequences from T-DNA, e.g. sequences encoding selectable markers including morphological abnormality induction genes. The removal of unwanted DNA fragments from T-DNA regions occurs either with the help of site-specific recombination (WO9742334; Endo et al., 2002, Plant J., 30, 115-122) or by means of transposition (U.S. Pat. No. 5,792,924).
Site-specific recombination has been used for removing auxiliary sequences from T-DNA regions. Although site-specific recombinase/integrase-mediated DNA excision is more efficient than integration, the selection for excision events is a necessity, which leads to an additional step of tissue culture or screening of progeny for desired recombination events. In summary, all processes of manipulation with T-DNAs stably integrated into plant chromosomes are time-consuming, unflexible, and in general restricted to simple excision (with less efficiency—to integration) of desired DNA fragments. In addition, these processes are usually very limited in combinatorial diversity, as they are restricted to simple manipulations with a limited number of known genes and regulatory elements.
Offringa et al. (EMBO J. (1990), 9, 3077-3084) have described an extrachromosomal homologous recombination event between two Agrobacterium-delivered T-DNAs in plant cells followed by integration of the recombination product into nuclear DNA. The extrachromosomal homologous recombination efficiency between the co-delivered T-DNAs in the plant cell was however too low to have practical applications for vector engineering in vivo and was therefore used as control experiment in scientific studies of the mechanism of homologous recombination in plants (Offringa et al., 1990, EMBO J., 9, 3077-3084; Tinland et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 8000-8004; Puchta et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 5055-5060). The frequency of homologous recombination followed by integration into chromosomal DNA was approximately 1% of the plant co-transformation frequency with two T-DNAs (Offringa et al., 1990, EMBO J., 9, 3077-3084; Tinland et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 8000-8004; Puchta et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 5055-5060). Due to the low overall efficiency of this process, practical applications of this method have not been developed.
The frequency of targeted integration of transiently delivered T-DNA into a pre-engineered loxP site in plants is also very low. For example, Vergunst and colleagues (1998, Nucl. Acids Res., 26, 2729-2734) demonstrated that the frequency of Cre-mediated site-specific integration of an Agrobacterium-delivered T-DNA fragment of interest into a genomic T-DNA region with a loxP site is within the range of 1.2-2.3% of the number of random integration events. Due to this low efficiency, such integration processes require an additional selection round and the use of tissue culture to recover the cells carrying recombination events. In contrast to that, the frequency of chromosomal double-stranded DNA rearrangements with the help of site-specific recombinases is significantly higher and occurs in 29-100% of all plant germ cells (Zuo et al., 2001, Nature Biotechnol., 19, 157-161; Luo et al., Plant J., 23, 423-430). This is not surprising, as site-specific integrases/recombinases require double stranded DNA substrate for recognition of recombination sites and performing the reaction of site-specific recombination (Panigrahi et al., 1992, Nucleic Acids Res., 20, 5927-5935; Martin et al., 2002, J. Mol. Biol., 19, 107-127; Thorpe et al., 2000, Mol. Microbiol., 38, 232-241).
All data mentioned above suggest that T-DNA transiently delivered into the plant cell is a poor substrate for site-specific recombinases.
In a previous invention, we have overcome the above-described low efficiency by site-specific recombination-mediated assembly of RNA-viral amplicons (WO02/088369). The assembled viral amplicons were capable of strong autonomous amplification, cell-to-cell and systemic movement and, therefore, could strongly amplify the rare recombination events. Said viral amplicons were assembled in planta from two or more vectors by recombinase-mediated site-specific recombination and contained a gene of interest to be expressed transiently with the aim of achieving the strongest possible expression of the gene of interest throughout a plant that was infected by said vectors. However, expression of gene of interest was transient; stable transformation of plant chromosomes for stable and inheritable expression of a gene of interest was not addressed.
For many applications, the methods described in WO02/088369 can, however, not be used due to the following problems: Amplification and spread of the viral amplicon leads to viral disease symptoms that compromise plant health. Therefore, these methods cannot be used for gene function determination (functional genomics) since disease symptoms frequently obscure the function of a gene to be determined or prevent expression of the function to be determined. Further, expression of a gene of interest from an amplicon gives rise to unnaturally high expression levels leading to phenotypes different from the natural phenotype of that gene, perhaps due to unnatural interactions with functions of native genes of that plant.
Therefore, it is an object of the invention to provide an efficient, rapid and highly versatile process for transforming a plant or plant cells on a chromosome, notably a nuclear chromosome, whereby genetically stable transgenic plants or plant cells may be produced. It is another object of the invention to provide a process of producing transgenic plants transformed on a chromosome, whereby (e.g. for reducing cloning work) the DNA sequences to be integrated in said chromosome can be engineered in planta. It is a further object to provide a process of stably transforming plants or plant cells on a chromosome with a DNA sequence of interest having toxic effects on bacteria normally used for cloning said DNA sequence of interest. It is another object of the invention to provide a process of genetic transformation of plant nuclear DNA, which allows for screening for an optimal expression unit of a gene of interest. It is a further object to provide a process of stably transforming plants or plant cells on a chromosome, whereby vectors can be used in a modular fashion, for reducing the cloning work and the overall size of the vector molecules. It is another object of the invention to provide a process of stably transforming plants or plant cells on a chromosome, whereby said process allows screening of DNA libraries for desired functions in plants. It is further object of the invention to provide an in planta process of shuffling genetic elements/gene fragments, whereby said process is linked with a process of stably transforming plants or plant cells with a DNA sequence of interest resulting from said shuffling.